High-flux anti-tumor drug screening cell model based on STAT3 and NF-kB two-signal channel serving as target, as well as building and application of high-flux anti-tumor drug screening cell model

A technology of anti-tumor drugs and cell models, which is applied in the construction of anti-tumor drug screening cell models and high-throughput anti-tumor drug screening cell models. It can solve the problems of incomplete simulation of tumor cells, increased system instability, and increased costs. problem, to achieve the effect of shortening the screening cycle, reducing operation steps and improving stability

Active Publication Date: 2012-06-13
上海中科生物医学高科技开发有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0003] At present, more and more researchers have realized the feasibility of using STAT3 and NF-kB as signaling pathways for anti-tumor drug screening, and have begun to try related methods, but The drug screening system based on STAT3 and NF-kB signaling pathway as the target, because the STAT3 or NF-kB activity of the selected cell line itself is relatively low, when screening specific inhibitors, it is often necessary to stimulate the cells with cytokines in advance, and then proceed For compound screening, the use of cytokines not only greatly increases the cost, but also increases the instability of the system with each additional step of the drug screening system; at the same time, the external stimulation cannot completely simulate the fact that tumor cells actually have higher STAT3 or NF-kB activity. state

Method used

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  • High-flux anti-tumor drug screening cell model based on STAT3 and NF-kB two-signal channel serving as target, as well as building and application of high-flux anti-tumor drug screening cell model
  • High-flux anti-tumor drug screening cell model based on STAT3 and NF-kB two-signal channel serving as target, as well as building and application of high-flux anti-tumor drug screening cell model
  • High-flux anti-tumor drug screening cell model based on STAT3 and NF-kB two-signal channel serving as target, as well as building and application of high-flux anti-tumor drug screening cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1. Construction of reporting system carrier

[0050] according to Figure 4 As shown in the schematic diagram, the response sequence is artificially constructed, which includes STAT3 binding sequence and NF-κB binding sequence. The STAT3 binding sequence is referred to as SIE, and the NF-κB binding sequence is referred to as κB. The STAT3 binding sequence here consists of 5'-TTCCCGTAA-3' and 5'-TTCCTGTAA-3' 8 alternating series; the NF-κB binding sequence is 5'-GGGAATTTCC-3'; the two binding sequences are connected in series, Then add the general TATA box auxiliary sequence 5'-TATATAA-3', insert it into the general luciferase reporter vector pBR322-luc-puro, and the recombinant vector is named SIE-κB-luc; as a control, STAT3 will be included alone Nucleic acid sequences of the binding sequence or NF-κB binding sequence (the rest are the same) are inserted into the luciferase reporter vector, named SIE-luc and κB-luc, respectively. The ratio of the two response elemen...

Embodiment 2

[0065] 1. Construction of reporting system carrier

[0066]①Use the SIE-κB-luc vector in Example 1 to extract with the Tiangen High Purity Plasmid Mini-Extraction Kit, take 10 μg for double enzyme digestion with XhoⅠ and BglⅡ (3 μl each), 37 °C, 8 h, Agarose gel electrophoresis, using the Tiangen DNA Purification and Recovery Kit to recover the product I, the product at this time excised the κB sequence; two DNAs with cohesive ends (and can be paired with XhoI and BglII restriction sites) Complementary primers, including two SIE sequences 5'-TTCCCGTAA-3' and two κB sequences 5'-GGGAATTTCC-3', see underlined)

[0067] 5'-TCGAGC GGAAATTCCCGTAA AC GGAAATTCCCGTAA AA-3', and

[0068] 5'-GATCTT TTACGGGAATTTCC GT TTACGGGAATTTCC GC-3'

[0069] Dilute to a 50 μM solution, mix 15 μl each, put in a boiling water bath at 95 °C for 5 minutes, and cool naturally in the water bath to obtain product II; Ligation, ligation overnight at 16°C; ③Transform Escherichia coli competent DH5...

Embodiment 3

[0081] 1. Construction of reporting system carrier

[0082] Using the same primer annealing and enzyme-cut ligation methods as in Example 1, the complete insert sequence inserted into the multiple cloning site of the luciferase reporter vector pBR322-luc-puro is as follows:

[0083]

[0084] 2. Report system carrier performance test

[0085] Same as Example 2, but when 293T cells are used to detect the simultaneous activation of STAT3 and NF-kB signaling pathways, the fluorescence value that can be achieved by the reporter system is only about 37% greater than the sum of activation of individual signaling pathways, and the fluorescence value increased by STAT3 activation is only Adding about 10% of the fluorescence value for NF-kB activation, the decrease in fluorescence value when using the same inhibitor to inhibit the STAT3 signaling pathway is not as obvious as the reporter system vectors in Examples 1 and 2.

[0086] 3. Drug screening model cell selection

[0087] Wi...

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Abstract

The invention provides a high-flux anti-tumor drug screening cell model based on an STAT3 and NF-kB two-signal channel serving as a target. In the cell model, the specific binding sequence of STAT3 and that of NF-kB are bound to build a reporting system carrier; and the cell model is built by binding competent cells comprising both constitutive STAT3 and constitutive NF-kB. The cell model is used for screening and treating cell cancerization caused by continuous activization of STAT3 and / or NF-kB, and medicines generated by the combination and the mutual promotion of STAT3 and NF-kB, and has a higher efficiency than a single-signal medicine screening system. As the competent cells comprising both constitutive STAT3 and constitutive NF-kB are adopted, the reporting gene in the cell model is in a highly active state, no stimulus is required to be added additionally, the screening process is simplified, the screening cost is reduced, the stability, the high efficiency and the easiness in the use of a system are improved, and the cell model is more suitable for high-flux medicine screening.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a cell model for screening anti-tumor drugs, in particular to a high-throughput cell model for screening anti-tumor drugs based on dual signaling pathways of STAT3 and NF-κB; Construction method and application of cell model for anti-tumor drug screening. Background technique [0002] In many tumor cells, especially in colon cancer, breast cancer, glioma cells, lung cancer and other cells, there are continuously activated STAT3 (signal transducer and activator of transcription 3) and NF-kB (nuclear factor-κB )active. The NF-κB family mainly includes the following members, specifically RelA (p65), RelB, c-Rel, p50, and p52. Usually, NF-κB exists in the form of homologous or heterodimer, among which p65 / p50 dimer Is the most common form of NF-κB in mammals. Under normal circumstances, NF-κB dimer is mainly retained in the cytoplasm due to the combination of its inhibitory protein IκB (I...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/63C12N15/11C12Q1/66C12Q1/02G01N21/64
CPCC12N5/10C12Q1/6897G01N33/5011C12N15/63C12N15/11C12Q2600/112
Inventor 杨金波王勤杜宇平陈星
Owner 上海中科生物医学高科技开发有限公司
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